Cryptosporidium parvum (C. parvum) is a food or waterborne parasite that infects humans and animals causing severe intestinal distress. Infection caused by C. parvum is particularly dangerous because it can cause prolonged diarrheal illness that may be potentially fatal for immunocompromised individuals. Since the 1970s, C. parvum has been receiving increased world wide attention as the frequency of outbreaks and the number of individuals infected increase across the globe. For example, in an outbreak reported in Milwaukee, Wis. in 1993, approximately 400,000 people were infected with C. parvum and 50 premature deaths were attributed to the infection. Outbreaks have also been reported in Las Vegas, Nev.; London, England; and Australia. Therefore, the U.S. Environmental Protection Agency has begun the process of mandating that waters in the United States be tested for Cryptosporidium. 
C. parvum oocysts are easily moved between watersheds by birds, and mammals, both domestic and wild. The remarkable resistance of oocysts to disinfectants, oocysts long-term environmental survival, and low infectious dose shows that conservative guidelines in detection and quality control of drinking water should be followed.
Although, cryptosporidiosis occurs worldwide, children, travelers to foreign countries, immunocompromised individuals and medical personnel caring for patients with the disease, are at particular risk. Apart from humans, Cryptosporidium infections are widespread in several other vertebrates including mammals, reptiles and fish. Cryptosporidium parvum in non-human mammals, but not reptiles or fish, is infectious to humans. Accordingly, the frequency of cryptosporidiosis in animal handlers and veterinarian personnel is reported to be relatively high.
Considerable efforts have been made to develop and improve Cryptosporidium detection methodologies through the application of a wide range of techniques such as flow cytometry, laser scanning, immunomagnetic separation, and polymerase chain reaction (PCR). However, the ability of existing detection methods to detect C. parvum in environmental samples has been limited due to factors such as interference caused by high sample turbidity and the inability to differentiate between viable and non-viable oocysts. Because the minimum infective dose is low (between 30-100 viable oocysts), the volume of sample to be analyzed is small, and the Cryptosporidium organism exists in several forms during its life cycle, detection methods must be highly sensitive or must utilize extensive sample concentration steps in order to be reliable.
The C. parvum life cycle is as follows: Oocysts enter the gastrointestinal system of the host, generally by the ingestion of contaminated food or water, and invade the intestinal and, very rarely, urogenital systems where the oocysts mature and release sporozoites. The sporozoites reproduce asexually to produce additional oocysts. These infective oocysts pass into the feces and are excreted. Following ingestion of the oocysts by another vertebrate, the oocysts release sporozoites that attach themselves to the epithelial surface of the gastrointestinal system and initiate a new cycle of infection by intercellular invasion.
As C. parvum organisms invade the surface of intestinal cells, the host experiences symptoms such as reduced appetite, severe diarrhea, abdominal cramping, and chronic fluid loss. The symptoms generally persist for five to eleven days, and then rapidly abate. However, in immunocompromised individuals, such as malnourished children, individuals with congenital hypogammaglobulinemia, those receiving immunosuppressants for cancer therapy or organ transplantation, and patients with AIDS, onset of the disease is more gradual and diarrhea is more severe, causing extreme fluid losses. Unless the underlying immunologic defect is corrected, the diarrhea may continue persistently or remittently for life because there is no effective, specific anti-C. parvum therapy available at present. Although some patients have responded positively to therapy with conventional antibiotics such as spiramycin and paromomycin, the result of infection is frequently fatal for immunocompromised individuals. In fact, cryptosporidiosis has been reported as one of the predominant causes of death in immunocompromised patients.
In light of the potentially fatal consequences of C. parvum infection, sensitive methods for detecting C. parvum contamination are necessary. In humans, the typical source of C. parvum is contaminated water, therefore the detection of Cryptosporidium in drinking and recreational water sources is a primary goal.
Currently available detection systems indicate that C. parvum organisms are observed in “spikes”; meaning that levels of C. parvum in samples collected upstream and downstream, from the same source of contamination, may not be identical when simultaneous readings are made. Consequently, C. parvum levels recorded from one location may differ significantly from readings taken from the same location minutes later. Detection of C. parvum in water is further complicated because the initial source of the infectious agent is difficult to identify. An abnormally high C. parvum concentration may be caused by water run-off from contaminated farm or pasture land, or an infected infant's soiled diaper carelessly discarded into a stream or worn in a public pool.
Ideally, continuous filtration systems having the capability to capture and retain C. parvum organisms for subsequent analysis would be installed in all water supply reservoirs to allow for continuous monitoring. Unfortunately, filtration systems currently in use often have filtration cartridges that either fail to retain organisms, frequently become clogged with mud or sediment, or must be replaced or cleaned with a frequency that renders the cartridges impractical.
C. parvum detection assays presently in use are cumbersome and frequently inaccurate. For example, most assay test samples begin as crude mixtures of C. parvum oocysts separated out from mud deposits collected by filters. The oocysts are isolated by processes involving centrifugation and ultrafiltration. Separating oocysts in this manner is often tedious and inefficient since each time the test sample is spun and filtered, oocysts are lost in the process, inevitably resulting in a lack of sensitivity and related inaccuracies. Another significant disadvantage of such assays is the large amount of time required for processing test samples. For example, in order to improve the optical properties of test samples for detection, oocysts must be stained. Typically, staining and subsequent detection procedures can take up to four days. Furthermore, samples can be tested only in small increments, and the sensitivity of most currently available assays is very low. Generally at least 50,000 C. parvum oocysts per milliliter must be present for a positive detection result. However, the minimum infective dose is low, between 30 and 100 oocysts. Therefore, C. parvum assays currently in use are generally inefficient, inaccurate and inconsistent.
Another barrier to effective Cryptosporidium screening concerns sample turbidity. The term “turbidity” refers specifically to the clarity or transparency of water and the effect that any suspended particles in the water may have on this clarity. Turbidity is determined by quantifying the amount of light allowed to pass through a sample and is measured in NTUs (nephelometric turbidity units). Many source water sites of public water reservoirs (e.g., rivers and lakes) often have turbidities up to 100 NTUs, whereas finished water (e.g., reservoirs for public consumption) tend to have turbidities in the range of 0 to 5 NTUs. High turbidities are defined herein as having greater than 10 NTUs.
Because it is commonly suspected that Cryptosporidium contamination occurs at source water sites, efforts have been focused on assaying samples at reservoir intakes. Several liters of source water are pumped through filters that are rated to capture particles the size of oocysts or larger. Pumping source water in this way causes large amounts of sediment to obstruct the flow of water through filters and therefore limit the volume of water passing through the filters. The filter retentates are then eluted and assayed for the presence of microorganisms. These retentates can have turbidities up to 300,000 NTU and yield highly variable C. parvum oocyst counts by immunofluorescence assay due to the loss of oocysts that occurs in multi-step sample processing. Concentrations of the retentates can increase turbidities further.
Oocysts present in filter eluate often tend to be washed away during processing and therefore go undetected in the final step of detection assays. Consequently, currently available methods such as immunofluorescence assays (IFA) and enzyme immunoassays (EIA), are mainly useful for detecting oocysts in “clean” samples (i.e., samples that have low turbidity). Such assays are more likely to give reproducible results with clean samples than those that are considered “dirty” (i.e., samples that have high turbidity).
Currently available Cryptosporidium detection methods for public health surveillance of oocyst exposure are incapable of distinguishing C. parvum from other Cryptosporidium species. In addition, current detection methods count the total number of oocysts in the sample, without regard for viability; therefore, both viable and non-viable oocysts are counted. Oocyst viability, measured by the ability of an oocyst to excyst, is valuable because over time, oocysts lose the ability to excyst and thus become noninfective. Therefore, any attempted correlation between the number of oocysts in drinking water and the incidence and risk for disease in healthy and immunocompromised persons is unreliable.
Clinical diagnosis of cryptosporidiosis is made by recovering acid-fast oocysts from stool samples. Excretion of acid-fast oocysts is most intense during the first four days of illness but persists for the duration of diarrhea. Other assays currently in use for diagnostic purposes involve the use of formalin-ethyl acetate sedimentation or Sheather's sugar flotation stool concentration procedure to enhance the yield of oocysts in specimens containing few oocysts. Commercial fluorescein-labeled monoclonal antibody kits also provide detection of oocysts in clinical specimens. (Merck Manual, Chapter 15 pp. 237-238 16th ed. (1992)). The disadvantage of such clinical tests is that, depending on the stage of C. parvum infection, the assays may or may not be adequately sensitive for detecting oocysts. In addition, such clinical tests generally involve a multitude of steps thereby introducing a greater likelihood of inaccuracies. Furthermore, no “standard” for testing stool specimens for C. parvum has been established, and so the absolute sensitivity of currently used methods has not been assessed. (Christine L. Roberts et al., JOURN. OF CLIN. MICRO., Vol. 34 No. 9, pp. 2292-2293 (1996)). Other problems associated with C. parvum testing include extensive processing time and low test positivity rates.
In summary, existing assays for C. parvum parasites are irreproducible, non-specific, insensitive, labor-intensive, susceptible to interference by sample turbidity, and time consuming. In addition, existing assays are not quantitative, lack the ability to distinguish different species of Cryptosporidium or distinguish infectious from non-infectious organisms, and are unable to correlate parasite levels in drinking water with incidence and severity of disease. Useful assays that enable correlation of disease-parasite levels are required for the development of environmental guidelines for safeguarding water sources against C. parvum and other parasitic infestation.
What is needed, therefore, is a sensitive, quantitative and reproducible assay for C. parvum. 